CD and NMR Studies on the Interaction of
Lithium Ion with Valinomycin and Gramicidin-S
M. 8.SANKARAM and K. R. K. EASWARAN,* Molecular Biophysics
Unit, Indian Institute of Science, Bangalore 560 012, India
Synopsis
The conformation of the valinomycin-lithium complex has been studied using CD and nmr
techniques. The lithium ion induced significant changes in the chemical shifts of the NH
and CaH protons. as well as in the CD spectra of valinomycin. From the analysis of the lithium
ion titration data,it is concluded that valinomycin forms a 1:l type weak complex with lithium,
having a stability constant of 48 L mol-l at 25OC. This conformation is different from the
familiar valinomycin-potassium complex. T h e nature of the interaction a t low and high
concentrations of lithium ions with valinomycin (ionophore) and gramicidin-S (nonionophore)
has been compared. At high salt concentrations, there was a further change in the C D and
nmr spectra of valinomycin, giving a second plateau region a t >3M of the salt. In the case
of gramicidin-S, no significant changes in the nmr or CD spectra were observed in the lower
concentration range corresponding to where changes were observed in the case of valinomycin.
However, the addition of lithium salt at concentrations greater than 3M induced changes in
both the CD and nmr spectra of gramicidin-S, and the titration graph of molar ellipticity versus
concentration of lithium perchlorate gave a plateau region at concentrations greater than this.
These results indicate that the effects of lithium a t low and high concentrations are independent of each other. The conformational transitions a t very high salt concentrations
(denaturation) are more likely due to solvent structural perturbations rather than to the
consequences of ion binding.
INTRODUCTION
The effects of alkali metal cations on the conformations of biological
macromolecules are of importance in understanding their functions. A t
low concentrations of salts, appropriate stoichiometric meta1:peptide
complexes may be formed. For example, the cation binding properties of
ionophoric peptides, depsipeptides, and polyethers are well documented.'
A t high concentrations of the salts, it is well known that proteins and
polypeptides are denatured.2 However, the question of whether, and how,
the site binding is related to denaturation does not seem to be very well
understood. An answer to this question is of interest, since both involve
conformational changes. One way of approaching such a problem would
be to compare the effects of the addition of salts at varying concentrations
* T o whom correspondence should be addressed.
Biopolymers, Vol. 21.1557-1567 (1982)
Q 1982 John Wiley & Sons,Inc.
CCC OOOS-3525/82/081557-11%02.10
SANKARAM AND EASWARAN
1558
on ionophoric and nonionophoric systems. We have chosen valinomycin'
and gramicidin-S3 for this purpose.
The ability of valinomycin to selectively transport potassium ions has
led to a detailed characterization of its complexes with the monovalent
alkali metal cations, Na+, K+, Rb+, and Cs+.' The complexation of the
lithium ion has always been considered weak, and no systematic investigations have been carried out to characterize it otherwise. Because Fthium
transport4 across biological membranes is not as clearly understood as
potassium and sodium transport, it is important to characterize the complex. Although valinomycin is known to be a potential ionophore for potassium acting through a carrier mechanism, the various steps of the
transport (the complexation and decomplexation at the interface, diffusion
across the membrane, etc.) have not been experimentally demonstrated.
In our laboratory, we are studying valinomycin and its complexes with a
variety of cations of varying size and charge to try to understand all the
possible conformations the complex can take. Recent investigations of
the valinomycin-barium system5v6have demonstrated the existence of 1:2.
1:1,and 2:l va1inomycin:barium complexes.
-1.2
1
205
I
215
I
225
I
235
I
245
h(nm1
Fig. 1. CD spectrum of (a) valinomycin and (b) valinomycin-lithium complex (150)in
acetonitrile; valinomycin concentration, 5 mM.
CD AND NMR OF LITHIUM COMPLEXES
-0.2
I
1559
I
I
I
I
25
50
75
100
b+I/ CVMI
Fig. 2. Plot of relative molar ellipticity versus [Li+]/[peptide]at 217 nm for valinomycin
(0)
and at 205 nm for gramicidin-S ( 0 ) . Concentrations: valinomycin, 4.87 mM; gramicidin-S, 0.13 mM.
In this paper, we report our investigations on the valinomycin-lithium
complex and the effects of high concentrations of lithium salt on the conformations of valinomycin and gramicidin-S, in the lipophilic solvent acetonitrile.
EXPERIMENTAL
Valinomycin and gramicidin-S were from Sigma Chemical Company.
Lithium perchlorate was purchased from Alfa Chemical Company, and
acetonitrile-da was from Stohler Isotope Chemicals. Acetonitrile used for
CD measurements was distilled after refluxing over P205 for 2-3 h. Solutions for the titrations were prepared by appropriate mixing of the stock
solutions of valinomycin and valinomycin plus lithium perchlorate in acetonitrile. The valinomycin concentration used for both CD and proton
nmr experiments was -5 mM. CD spectra were recorded on the Jasco 5-20
spectropolarimeter. 'H-and 13C-nmrexperiments were done on a Bruker
WH-270 Fourier-transform nmr instrument equipped with a variabletemperature accessory. Spectra were obtained in the Fourier-transform
mode and were generally the result of 25-50 accumulations for 'H-nmr and
150-200 for 13C-nmr. Chemical shifts are expressed ppm downfield from
TMS as the internal reference. AU experiments were done at a temperature
of 25OC.
SANKARAM AND EASWARAN
1560
RESULTS
CD Studies
The CD spectra of valinomycin in acetonitrile (Fig. 1) showed striking
changes on addition of lithium perchlorate salt. On the gradual addition
of salt, a new band at 217 nm appeared, with a negative molar ellipticity,
which increased to a value of -10.3 X lo3 deg cm2 dmol-'. The plot of the
relative molar ellipticity versus [metal]/[valinomycin] is shown in Fig. 2.
The actual [metal]/[valinomycin] ratios do not correspond to the stoichiometry of the complex, possibly because of the weak binding of lithium to
valinomycin and the strong solvation of the lithium ion. It is worth noting
here that the cation solvation Gibbs free energies of the lithium ion in acetonitrile7 and waterS are 114.5 and 122 kcal/mol, respectively. The titration graph shown in Fig. 2 gives a plateau region for the [metal]/[vaiinomycin] ratio of 50-100. However, the addition of salt a t high concentrations (0.5-5M lithium perchlorate) results in a decrease in the magnitude
of the molar ellipticity at 217 nm. The plot of the relative molar ellipticity
versus the lithium perchlorate concentration for the high concentration
range is given in Fig. 3.
In the case of gramicidin-S, no CD spectral changes were observed even
at the [lithium]/[valinomycin] ratio of 100 (Fig. 2). The spectral changes
were observed only at high salt concentration. The CD spectra of gramicidin-S in acetonitrile, containing a minimum amount of methanol, with
no salt, and with 3.5M lithium perchlorate are shown in Fig. 4. The solu-
0.3
-
-
*
I-
E
u
0.2
-
E
m
0
n
U
m
4
0
1
3
2
L
[LI CLOLl
Fig. 3. Plot of relative molar ellipticity of valinomycin (at 217 nm) versus concentration
of lithium perchlorate in mol L-I. The bracketed region in the curve is shown in Fig. 2.
Concentration of valinomycin was 4.87 mh4.
CD AND NMR OF LITHIUM COMPLEXES
-281
200
,
,
,
,
,
1
210
220
230
240
250
260
1561
A(nm)
Fig. 4. CD spectra of (a) gramicidin-S and (b)gramicidin-Swith 3.5M lithium perchlorate
in the solvent mixture of acetonitrile/methanol,101. Concentration of gramicidin-S, 0.13
mu.
bility of gramicidin-S in acetonitrile is limited; hence, a minimum amount
of methanol is used to solubilize gramicidin-S in acetonitrile. This means
that methanol forms a solvent sheath around gramicidin-S, and the bulk
is composed of acetonitrile. As seen in the figure, the CD curve at this salt
concentration is distinctly different from that of gramicidin-S with no salt.
No appreciable changes in the CD spectra of gramicidin-S were observed
on addition of other mono- and divalent metal ions such as Na+, K+, Ag+,
and Mg2+. However, Ca2+,Ba2+and Sr2+decrease the molar ellipticity
of gramicidin-S but do not give the same type of CD curve as that with
lithium. The plot of relative molar ellipticity versus lithium perchlorate
concentration for gramicidin-S is given in Fig. 5.
NMR Studies
The 270-MHz 'H-nmr spectrum of free valinomycin and valinomycinlithium complex (at the [valinomycin]:[lithium] ratio of 1:80)is given in
Fig. 6. The spectrum of the valinomycin-potassium complex is also inserted for comparison. The assignments of the various signals of the free
valinomycin in CD&N is the same as reported by Ovchinnikov and I v a n ~ v . ~
The assignments of the signals for the complex were made by carefully
examining the spectra at each point of the titration. The chemical shift
and spin-spin coupling constant data for the free valinomycin and its
lithium and potassium complexes are listed in Table I. The salt-induced
v
chemical shifts of the Ca protons of L-Lac, D-Val, L-Val, and D - H ~ Iresi-
SANKARAM AND EASWARAN
1562
N
5
12
dr
0
-r
0.5
[LI
1.5
2.5
3.5
5.5
4.5
ClO&]
Fig. 5. Plot of carbonyl carbon chemical shift of one of the intramolecularly hydrogenbonded carbonyls of gramicidin-S ( A ) and relative molar ellipticity (0)at 205 nm vs
[LiCIOJ].
dues and the NH proton of the valine residues versus [metal]/[peptide]
ratios are shown in Fig. 7, where the salt-induced chemical shifts are different for the different protons (for example, the maximum shift for CaH
protons of L - V is
~ 0.02 ppm as compared to 0.14 ppm for the C"H proton
of D-Val). However, as observed in the CD spectrum, the titration graph
showed a plateau region for the chemical-shift variation in the concentration range studied. The temperature coefficient of chemical shift of the
I
I
8.5
8.0
7.5
"5.5
5.0
6 (ppm)
I
I
I
L.5
L.0
3.5
Fig. 6. The 2 7 0 - m proton
~
spectra of (a) valinomycin, (b)valinomycin-potassium complex
(1:l).and (c) valinomycin-lithium complex (1230) in acetronitrile-ds. Concentration of
valinomycin, -5 m M .
8.42
7.46
7.60
8.34
D-Val
4.17
3.85
4.19
4.13
3.80
4.27
5.24
4.95
5.07
Chemical Shift (ppm)
C-H
I.-Val
D-Val
L-Lac
4.98
4.64
4.79
D-HyIv
.
7.50
4.17
6.60
I.-Val
JNHOH'
8.40
4.77
6.20
D-Val
9.80
11.00
5.50
l.-Val
8.70
I1.OO
4.10
D-Val
JC-HCW
Coupling Constant, J (Hz)
Measurements made at the valinomycin-cation ratio of 1:l for potassium complex and 1:80 for lithium complex. Temperature, 25°C
All chemical shifts measured with respect to Me& as internal standard.
c Correction factor for electronegativity not included.
Data taken from Ref. 9.
Valinomycin
Valinomycin t Kt
Valinomycin t Lit
[.-Val
NH
3.80
3.60
5.10
D-HYIV
TABLE I
Proton Chemical Shifts and Coupling Constants for Free Valinomycin and Its Complexess with Lithium and Potassium Salt in Acetonitrileb
SANKARAM AND EASWARAN
1564
0.16 I
0-
0.12
0.08
0.04
-
0
,a
ln
a
-0.04
E
-0.08
-0.12
-0.16
-0.20
Fig. 7. Titration graph of L-Val(O),&Val (e),
D-HYIV( A ) , L-Lac (0)
Ca protons and NH
protons (A)of valinornycin vs [Li+]/[valinomycin] ratio. Concentration of valinomycin, 5.4
mM.
NH protons of L-Val and D-Val at a peptide:metal ratio (1:80) in the plateau
region of Fig. 7 is -2.1 ppb/"C. A comparison of this value with that of
valinomycin in acetonitrile (-3.9 ppb/"C for L-Val and -3.6 ppb/"C for
D-Val, as well as in other solventslO),indicates that the hydrogen bonds
are intact and are probably strengthened during complexation. The
structural characteristics at very high salt concentrations could not be
studied because of the broadening of the nmr spectra.
In the case of gramicidin-S, the changes on addition of lithium perchlorate were monitored by l3C-nmr. The assignment of carbonyl signals for
gramicidin-S in this solvent system has not been done. However, of all the
carbonyl signals, the one observed at the lowest field (172.21 ppm) was
monitored, since this shows a continuous upfield shift as compared to
others, which overlap. It is possible that this signal can be assigned to the
carbonyl of one of the following residues": proline, D-phenylalanine, or
leucine; the carbonyl of the last residue is involved in intramolecular hydrogen bonding. No changes in the 13C chemical shift were observed in
the concentration range where valinomycin binds lithium. A t high salt
concentration ( > 3 M ) , the carbonyl carbon showed an upfield shift. The
salt-induced chemical shift of the carbonyl carbon (the 172.21 ppm signal
of gramicidin-S) is plotted as a function of salt concentration in Fig. 5.
CD AND NMR OF LITHIUM COMPLEXES
1565
DISCUSSION
The CD and nmr results of the effect of lithium salt on valinomycin and
gramicidin-S clearly show that the nature of the interaction of the salt is
different in the two cases. In the case of valinomycin, the two plateau regions in the titration graphs, one at low (Fig. 2) and the other at high (Fig.
3) salt concentrations, indicate two different processes or conformations
for the molecule at these concentrations. In the case of gramicidin-S, the
effect on the CD and nmr spectra is observed only at high lithium salt
concentrations. The effects observed at high salt concentration for both
valinomycin and gramicidin-S could only be due to similar interactions
affecting the structural stability of the peptide. The changes in the spectral
parameters at low salt concentrations (<0.5M salt), only in the case of
valinomycin, are due to the binding of the lithium ion to valinomycin. The
absence of such spectral changes for gramicidin-Sat low salt concentrations
(<0.5M;Fig. 2) indicates only that gramicidin-S does not bind the lithium
ion.
Complexation of Lithium by Valinomycin
Analysis of the CD data by the method of Rose and Henkens12showed
that the valinomycin-lithium complex has a 1:l stoichiometry with weak
binding, as shown by the stability constant obtained, i.e., 48.1 f 0.5 L mol-l.
The data do not fit any equilibrium with other stoichiometry or any multiple equilibria-this has been checked by Reuben'sL3method.
The 'H-nmr titration graph showed very different behavior for the ~ - V a l
and D-Val CaH proton salt-induced chemical shift, as compared to the
valinomycin-potassium complex.1 In the latter case, both these Ca protons
shift upfield on addition of potassium salt, whereas in the case of the valil proton chemical shift remains unnomycin-lithium system, the ~ - V aCa
changed and the D - V Ca
~ proton resonance shifts downfield (Fig. 7). This
indicates that the valinomycin-lithium complex is not of the valinomycin-potassium type; the complex possibly involves only one set of three
carbonyls (D-Val carbonyls, because the CaH of D-Val showed a large
change in chemical shift on complexation)as ligands. The lithium ion, with
its large hydration sheath possibly favors the L-Lac, ~ - V a side
l
of the
l D-HYIVresidues, both
bracelet, since the other side contains ~ - V a and
having bulky side chains. The continuous upfield shift of the D-HYIVmay
be due to its carbonyl carbon orienting inside the bracelet, strengthening
the intramolecular hydrogen bonds. This is consistent with the temperl protons
ature coefficient of chemical shift data for L-Val and ~ - V aNH
(for both AWAT = -2.1 ppb/"C at a valinomycin:lithium ratio of 1230).
The 3JNH-CmH coupling constants of the L-Val residues are 6.6 and 6.2 Hz,
respectively, and these values are consistent with the proposed model for
the complex, namely, a 1:l type with the lithium ion binding to the three
ester carbonyls of the &Val residue; the remaining ligands for the ion are
possibly from the solvent acetonitrile. The complex retains a CS sym-
1566
SANKARAM AND EASWARAN
metrical bracelet-type structure similar to the valinomycin-potassium
complex.
Although all IA group metal'ions are known to form complexes of the
valinomycin-potassium type, lithium, according to this study, forms a very
different type. This conformation, proposed for the valinomycin-lithium
complex, where the ion is bound to one side of the bracelet, is a direct
demonstration of the possible complexation reaction at the membranewater interface.
Effect of High Salt Concentrations
Lithium has been shown to bind to the amide carbonyl group of amides,lPl7 as well as to various charged groups that may be available on the
peptides.2 The binding of lithium to amide carbonyls is drastically affected
by the presence of even small amounts of water, and this has to be taken
into account when denaturation data at high salt concentrations are interpreted. Denaturation of proteins and polypeptides involves disruption
of intramolecular hydrogen bonds. A t high salt concentrations, the conformational transitions exhibited could be due either to ion binding or to
solvent structural changes.18J9
Figures 3 and 5 show a sudden conformational transition at high lithium
concentration ( > 3 M ) for both valinomycin and gramicidin-S, and this is
possibly due to breaking of the structure at such high salt concentrations.
The upfield shift of the carbonyl carbon chemical shift is consistent with
this. As we have already discussed, at low salt concentrations the lithium
ion binds to valinomycin, forming a 1:1type complex, whereas it does not
bind to gramicidin-S. This suggests that ion binding and denaturation
are independent of each other and that the conformational transition at
high salt concentrations is not a consequence of ion binding. Lithium, a
highly solvating ion, removes the solvent sheath around the depsipetide,
and this occurs at high salt concentration. The loss in the enthalpy of
solvation of the depsipeptide is made up, in part, by an increase in the entropy accompanying the structure breaking. Also, if ion binding assisted
in the structure breaking, we would expect divalent metal ions to be more
effective than monovalent ions. The fact that the effectivenessof the metal
ions follows the Hofmeister series18 can be interpreted as being due to
solvent structural changes. We believe, therefore, that, whether ionophoric
or not, the conformational transitions at high salt concentrations are not
driven by ion binding. Any net binding of lithium is due t o binding to the
carbonyls exposed by structure breaking.
This work was supported, in part, by a Department of Science and Technology, Government
of India, research grant for conformationalstudies on ionophores. The high-field nmr experiments were performed at the Bangalore NMR Facility of the Indian Institute of Science,
Bangalore.
CD AND NMR OF LITHIUM COMPLEXES
1567
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Received September 25,1981
Accepted January 29,1982